UMLS:C0004135 - FACTA Search

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Query: UMLS:C0004135 (ATM)
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This study addressed the potential radiosensitizing and DNA-damaging actions of the DNA topoisomerase I poison camptothecin (CPT) on SV40 transformed normal (MRC5CVI) and ataxia-telangiectasia (AT5BIVA) fibroblast cell lines. In both cell lines CPT induced a dose-dependent delay of cells in S phase, followed by a dose-dependent trapping in G2/M phase. Acute X-irradiation produced patterns of G2/M arrest and S-phase delay similar to those observed for CPT in the MRC5CVI cell line, but no S phase delay was observed in the AT5BIVA cell line consistent with the ataxiatelangiectasia phenotype of this cell line. X-irradiation of CPT-treated cells resulted in additive prolongation of S phase delay in MRC5CVI cultures and additive effects for cell killing in both cell lines. The potential for topoisomerase I-DNA cross-linking by CPT was not altered by 24h pretreatment with CPT, or by acute X-irradiation. Hypersensitivity of AT5BIVA to CPT was not attributable to elevated levels of complex trapping. These findings suggest that in a rapidly proliferating human tumour there is unlikely to be synergistic therapeutic gain when the two agents are used concurrently, and that previously reported radiosensitization by CPT is restricted to G0 phase cells.
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PMID:DNA damaging and cell cycle effects of the topoisomerase I poison camptothecin in irradiated human cells. 135 23

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.
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PMID:Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes. 164 93

Induction and rejoining of DNA single-strand breaks (ssb) and double-strand breaks (dsb) after gamma-irradiation were measured, respectively, by alkaline and neutral sucrose gradient sedimentation methods. The radiosensitive mutants irs1, irs2, and irs3 showed no significant difference from wild-type V79 hamster cells in ability to rejoin either ssb or dsb, while the previously-described xrs-1 mutant showed the expected defect in rejoining dsb. The resistance of DNA synthesis to gamma-irradiation was measured in the 3 irs mutants and, for comparative purposes, in transformed human cell lines from normal and ataxia-telangiectasia (A-T) individuals. The irs2 mutant was found to be very similar in response to the A-T lines, showing a marked decrease in inhibition of DNA synthesis, compared to V79 cells, in both time-course and dose-response experiments. However, irs1 also had some decrease in inhibition at the higher doses used, while irs3 was similar to the wild-type V79 cells. Both irs1 and irs2 were found to be considerably more sensitive to the DNA topoisomerase I-inhibitor camptothecin, while irs3 was only slightly more sensitive than the parent V79 line. These data place the irs mutants in a similar category of radiosensitive phenotype to A-T cells, but we view this as only the beginning of a useful classification of this type of mutant. The irs2 mutant has the strongest links to A-T cells, through its sensitivity profile to DNA-damaging agents and radioresistant DNA synthesis, but irs1 in particular has other similarities to A-T.
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PMID:DNA-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia-telangiectasia cells. 230 92

Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome.
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PMID:Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells. 283 4

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (gammaH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of gammaH2AX with: a. cell cycle phase; b. caspase-3 activation; and c. apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of gammaH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of gammaH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of gammaH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in gammaH2AX was apparent at a concentration range between 0.4 and 1.6 microM TPT. Thus, the intensity of gammaH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in gammaH2AX immunofluorescence. The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G1 or G2/M cells.
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PMID:DNA damage induced by DNA topoisomerase I- and topoisomerase II-inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis. 1450 78

Camptothecin (CPT) is a potent inhibitor of DNA topoisomerase I with a wide spectrum of anti-tumor activity. Relatively little information is available regarding the relation of known topoisomerase-mediated DNA damage with other intracellular pathways. To gain an insight into the intracellular molecular mechanisms of Topoisomerase I inhibitor camptothecin-mediated DNA damage leading to cell death, we used a high-density cDNA microarray to assess sensitive early gene expression profiles in SGC7901 (gastric cancer), Hela (cervical adenocarcinoma), K562 (chronic myelogenous leukemia) and HL60 (promyelocytic leukemia) tumor cells stimulated with camptothecin for 1 h at the concentrations of GI50 (50 % growth inhibition after 24 h of treatment). Analysis of the differentially expressed genes obtained 29 response genes common to all four cell lines. Moreover, these cell lines also shared the direction of regulation. Most of these common response genes were functionally related to cell proliferation or apoptosis, and some of them were involved in ATM (ataxia-telangiectasia mutated) and ATR (ATM-and Rad3 related) checkpoint pathways, JNK (c-Jun N-terminal kinase) pathway, the survival phosphatidylinositol (PI) 3 kinase-Akt-dependent pathway, mitochondrial cell death pathway, endoplasmic reticulum (ER)-related cell death pathway, and to ubiquitin/proteasome dependent protein degradation pathway. The data provides evidence for a linkage between topoisomerase-mediated DNA damage and intracellular signaling events, which may facilitate our understanding of the camptothecin mediated molecular mechanisms of action.
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PMID:Analysis of common gene expression patterns in four human tumor cell lines exposed to camptothecin using cDNA microarray: identification of topoisomerase-mediated DNA damage response pathways. 1636 68

Cellular response to DNA damage is complex and relies on the simultaneous activation of different networks. It involves DNA damage recognition, repair, and induction of signalling cascades leading to cell cycle checkpoint activation, apoptosis, and stress related responses. The fate of damaged cells depends on the balance between pro- and antiapoptotic signals. In this decisive life or death choice, the transcription factor NF-kappaB has emerged as a prosurvival actor in most cell types. As corollary, it appears to be associated with tumorigenic process and resistance to therapeutic strategies as it protects cancerous cells from death. In this review, we will focus on NF-kappaB activation by double-strand breaks inducing agents, such as ionizing radiation and DNA topoisomerase I and II inhibitors routinely used in cancer therapy. Coinciding with the 20th anniversary of the NF-kappaB discovery, major steps of the DSB-triggered cascade have been recently identified. Two parallel cascades are necessary for NF-kappaB activation. The first one depends on ATM (activated by double-strand breaks) and the second on PIDD (activated by an unknown stress signal). The phosphorylation of NEMO by ATM is the point of convergence of these two cascades. The identification of ATM/NEMO complex as the long searched "nuclear to cytoplasm" signal leading to IKK activation is also a major piece of the puzzle. The knowledge of the precise steps leading to DSB-initiated NF-kappaB activation will allow the development of specific blocking compounds reducing its prosurvival function.
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PMID:NF-kappaB activation by double-strand breaks. 1696 65

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitors doxorubicin, etoposide and mitoxantrone (MXT) are widely used antitumor drugs. They stabilize otherwise transient ("cleavable") complexes of topo1 or topo2 with DNA, respectively. Collisions of DNA replication forks (during replication) or progressing RNA polymerase molecules (during transcription) with these complexes convert them into double-strand DNA breaks (DSBs). Formation of DSBs triggers activation of ATM and phosphorylation of histone H2AX, the markers that have been used to correlate DNA damage with cell cycle phase or induction of apoptosis. In the present study we explored a relationship between H2AX phosphorylation and activation of checkpoint kinase 2 (Chk2) in human lung carcinoma A549 cells treated with TPT or with MXT. Activation of Chk2 was detected immunocytochemically using a phospho-specific (Thr68) Ab and measuring Chk2-Thr68(P)immunofluorescence (IF), concurrently with DNA content, by laser scanning cytometry. In the untreated cells, activated Chk2 was present predominantly in centrosomes. Upon treatment with TPT or MTX, the activated Chk2 presented itself in form of either minute or large IF foci in the cell's nucleoplasm. H2AX phosphorylation whether induced by TPT or MXT was rapid, with the maximal rate occurring during the initial 2 h and peaking at 2 h of treatment. TPT or MXT induced Chk2 activation occurred at a distinctly slower pace, peaking at 4 h. While TPT-induced H2AX phosphorylation and Chk2 activation were maximal in S-phase cells, Chk2 activation was also much pronounced in G(2)M cells; the least affected by TPT were G(1) cells. MTX-induced H2AX phosphorylation was maximal in G(1) cells while Chk2 activation was maximal in G(2)M and minimal in G(1) cells. The pattern of cell-cycle phase specific response to TPT or MXT by H2AX phosphorylation and Chk2 activation was different when measured either as integrated or maximal pixel of gammaH2AX or Chk2-Thr68(P) IF, the former reflecting total IF per nucleus the latter stressing the punctate (foci) character of expression of these phospho-modified proteins.
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PMID:Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase. 1845 60

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15(P)) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT-induced p53-Ser15(P) with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15(P) had "patchy" localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15(P) cytoplasmic immunofluorescence (IF). The intense phosphorylation of p53-Ser15, combined with activation of ATM and Chk2 (involving centrioles) as well as phosphorylation of H2AX seen in the untreated mitotic cells, suggest mobilization of the DNA damage detection/repair machinery in controlling cytokinesis. In the nuclei of cells treated with TPT or MXT, the expression of p53-Ser15(P) appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15(P) was most pronounced in S-phase cells while no significant cell cycle phase differences were seen in cells treated with MXT. The maximal increase in p53-Ser15(P) expression, rising up to 2.5-fold above the level of its constitutive expression, was observed in cells treated with TPT or MXT for 4-6 h. This maximum expression of p53-Ser15(P) coincided in time with the peak of Chk2 activation but not with ATM activation and H2AX phosphorylation, both of which crested 1-2 h after the treatment with TPT or MXT. The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by TPT or MXT, Chk2 rather than ATM mediates p53 phosphorylation.
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PMID:Phosphorylation of p53 on Ser15 during cell cycle caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation. 1880 8

Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is mediated by one of the protein kinases of the phosphoinositide kinase family, namely ATM, ATR, and/or DNA-PK. The presence of Ser-139 phosphorylated H2AX (gammaH2AX) is thus a reporter of DNA damage. This protocol describes quantitative assessment of gammaH2AX detected immunocytochemically in individual cells combined with quantification of cellular DNA content by cytometry. The bivariate analysis of gammaH2AX expression versus DNA content allows one to correlate DNA damage with the cell cycle phase or DNA ploidy. The protocol can also be used to assess activation (Ser-1981 phosphorylation) of ATM; this event also revealing DNA damage induced by topo I or topo II inhibitors. Examples where DNA damage was induced by topotecan (topo I) and etoposide (topo II) inhibitors are provided.
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PMID:Cytometric assessment of DNA damage induced by DNA topoisomerase inhibitors. 1976 48

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